The laboratory has been pursuing several related lines of research: 1. Characterization of PC1: The PKD1 gene encodes a 4302 aa, multi-transmembrane (TM) spanning protein, PC1. Previous studies by the lab have shown that PC1 undergoes auto-cleavage at the GPS site, which is located just before the first TM domain. After cleavage, the N-terminal fragment (NTF, 3000aa) is thought to remain tethered to the 11 TM C-terminus (1200aa) and the molecules are thought to traffic together as a complex to the plasma membrane. Several groups have reported that the intracellular carboxyl terminal fragment is subsequently released as a result of regulated intramembrane proteolysis (RIP) and then relocates to the nucleus where it functions to regulate gene expression. We have not been able to convincingly detect the RIP product. In order to better define the trafficking patterns of the full length protein and its proteolytic sub-components, we have generated new epitope-tagged PC1 constructs that allow us to detect both the N- and C-termini more reliably. We also have acquired live-cell, spinning disk capability in addition to standard LCM confocal access and have begun working out conditions that allow both static and dynamic, spatial imaging of PC1 and relevant co-factors in a wide variety of conditions (sub-confluent, confluent monolayer, when ciliated, and in 3D collagen gels). We believe this approach ultimately will yield important novel insights. In parallel efforts, we have continued to develop and characterize novel cell lines that can be used to study tubular morphogenesis. As outlined in last years report, we had acquired a spontaneously immortalized mouse renal collecting duct cell line from Switzerland, isolated subclones that reproducibly generate unambiguous tubules when cultured in matrix, and have examined the effects of silencing Pkd genes. As a complementary approach, we have generated several renal epithelial cell lines from Pkd1cond/cond, Immorto+, Cre- mice and initiated their characterization. We have shown that they produce elaborate tubules when cultured in matrix gels, and efforts are presently underway to optimize Pkd1 deletion. Finally, we have obtained the complete TALEN plasmid system from Addgene and begun testing several targeting constructs. We believe that this multi-tiered approach will both produce critical, robustly validated reagents for the PKD community and unique opportunities to study the mechanisms and regulatory pathways that govern tube formation. The studies are a natural complement to and extension of our in vivo work, described in the PKD mouse models section. Finally, we have been using the new PKD1 constructs, cell lines and imaging capacity to examine the functional relationship between PC1 and various small GTP-binding proteins. 2. PKD mouse models: Much of our effort continues to be focused on studying the earliest stages of cyst formation in mice. Using a mouse model of ADPKD carrying floxed Pkd1 alleles and an inducible Cre recombinase, we intensively analyzed the relationship between renal maturation and cyst formation by applying transcriptomics and metabolomics to follow disease progression in a large number of animals induced before P10. Weighted gene co-expression network analysis suggests that Pkd1-cystogenesis does not cause developmental arrest and occurs in the context of gene networks similar to those that regulate/maintain normal kidney morphology/function. Knowledge-based Ingenuity Pathway Analysis (IPA) software identifies HNF4a as a likely network node. These results are further supported by a meta-analysis of 1114 published gene expression arrays in Pkd1 wild-type tissues. These analyses also predict metabolic pathways are key elements in postnatal kidney maturation and early steps of cyst formation. Consistent with these findings, urinary metabolomic studies show that Pkd1 cystic mutants have a distinct profile of excreted metabolites, with pathway analysis suggesting altered activity in several metabolic pathways. To evaluate their role in disease, metabolic networks were perturbed by inactivating Hnf4a and Pkd1. The Pkd1/Hnf4a double mutants had significantly more cystic kidneys thus indicating that metabolic pathways could play a role in Pkd1-cystogenesis. A manuscript describing these findings is currently in press. In related work, we are perturbing other network nodes and determining how these interventions affect cyst formation and growth. We have also greatly expanded last years studies to include mice that develop the adult-onset form of the disease. In this model, we induce Pkd1 deletion at P40 and allow them to age for variable periods of time. We have collected serial urine samples and harvested tissues for microarray and histopathology at various intervals. The microarray studies have recently been completed on a large number of samples and the analyses are currently underway. In other studies, we have tested various induction strategies using the tet-inducible Cre recombinase line. Our preliminary studies suggest that we may be able to induce waves of somatic inactivation in a model that more closely approximates what happens in humans, but we have not found the tetracycline-inducible system to be as robust as had been reported in the literature. Likewise, some of our new reporter gene studies have yielded ambiguous results. Nonetheless, we have a number of striking findings that we are confident offer unique insights into the disease process, and we will continue to look for mechanistic explanations for these observations. Finally, we have made significant progress with our collaborators in characterizing a novel Pkhd1 mouse model. As noted in last years report, we had generated a new mouse line that has multiple copies of the HA epitope tag knocked-in to the extreme 3 end of the coding sequence of exon 67 and lox P sites flanking this exon. The newly introduced sequence appears to have no ill effects as homozygotes for the targeted allele are healthy. We have confirmed that the HA tag is expressed and detectable by both immunoblot and by immunstaining. We also have induced germline deletion of exon 67 and examined the phenotype of homozygotes up to 8 months of age. The results are notable for the apparent absence of either kidney or liver histopathologic abnormalities. This is the first mouse Pkhd1 mouse model that lacks any obvious phenotype. This is an unanticipated finding and will require further analysis to determine why loss of what was predicted to be an essential domain of the protein had no ill effects. These findings will be presented in an oral communication at this years ASN. Publications in Press: Network Analysis of a Pkd1-mouse model of autosomal dominant polycystic kidney disease identifies HNF4&#945; as a disease modifier. Menezes LF, Zhou F, Patterson AD, Piontek KB, Krausz KW, Gonzalez FJ, Germino GG. PLosGenetics, in press.